This retrospective study evaluated the diagnostic accuracy of F-18 FDG PET/CT with optimal cut-off values of the maximum standardized uptake value (SUVn = 118) and large  LN groups. The optimal cut-off value of SUVmax was determined for each group, using receiver operating characteristic curve analysis. Areas under the curve (AUC) were compared by C-statistics using two methods: the cut-off value of SUVmax optimized according to LN size, and a fixed SUVmax cut-off value of 2.5.This study included 176 patients with rectal cancer who underwent F-18 FDG PET/CT for initial staging. Patients were classified based on the long-axis diameter of the regional LN on CT images as small  in the small LN group, but not in the large LN group .The optimal cut-off values of SUVmax improves the diagnostic performance of F-18 FDG PET/CT for the evaluation of small regional LNs in patients with rectal cancer.Application of the lower cut-off value of SUVThe online version of this article (10.1186/s40644-018-0165-5) contains supplementary material, which is available to authorized users. Globally, colorectal cancer is the second most common cancer in women and the third most common cancer in men . In KoreConventional computed tomography (CT) and magnetic resonance imaging (MRI) have been commonly used for LN staging in patients with rectal cancer. However, both CT and MRI are limited by low sensitivity in the evaluation of small metastatic LNs –8. Recenmax) in the diagnosis of LN metastasis, without considering the size differences of the LNs. Application of optimal SUVmax cut-off values according to LN size may improve the sensitivity of F-18 FDG PET/CT and may be practically useful for evaluation of the regional LNs in patients with rectal cancer. Thus, the aim of this study was to evaluate the diagnostic accuracy of F-18 FDG PET/CT using optimal SUVmax cut-off values according to LN size to evaluate regional LNs in patients with rectal cancer.Several methods have been developed to correct the partial volume effect, and have significantly improved the diagnostic accuracy of metastatic LNs , 18. Hown = 118) and large  LN groups. The reference value for long-axis diameter was determined as 7 mm, because the partial volume effect is significant when the target of interest is smaller than 2 times of the PET/CT system’s full-width at half-maximum (FWHM) (< 8 mm) [We retrospectively analyzed the medical records of patients who underwent preoperative F-18 FDG PET/CT followed by curative operations for rectal cancer at our institution between January 2009 and August 2016. We excluded patients who underwent preoperative chemoradiation therapy and those with an interval of > 4 weeks between F-18 FDG PET/CT and surgery. A retrospective cross-sectional analysis was performed to review the surgical and pathological findings and the F-18 FDG PET/CT results. Patients were classified based on the long-axis diameter of the regional LN on CT images as small  , 20. ThiAll surgeries were performed by qualified, experienced colorectal surgeons. Mesorectal excisions were performed in all patients; extended LN dissections were only performed if metastatic LNs were detected in frozen biopsies. All resected LNs underwent histopathologic exams for pathologic confirmation while labeling the exact location. The sensitivity, specificity, and accuracy of F-18 FDG PET/CT were calculated using the histopathologic result as the gold standard. A true positive was defined as a match between the location of the metastatic LN on pathologic examination and the location of the positive LN on an F-18 FDG PET/CT image.Two different F-18 FDG PET/CT systems were used . The patients were required to fast for > 6 h before the scan, and the blood glucose level was measured to confirm that the level was < 180 mg/dL before injecting the F-18 FDG. In patients with diabetes, administration of antihyperglycemic drugs was stopped 12 h before the scan. Patients received intravenous administration of 4.0 MBq/kg (Biograph mCT) and 7.0 MBq/kg (Discovery STE) F-18 FDG according to the PET/CT system. Patients were encouraged to rest during the F-18 FDG uptake period. Images were acquired 60 min after F-18 FDG administration. A non-contrast CT scan was obtained for attenuation correction and localization. Immediately after the CT scan, PET images were acquired from the base of the skull or top of the brain to the proximal thigh. The Discovery STE-16 PET/CT scanner acquired images with a slice thickness of 3.75 mm simultaneously for a longitudinal field of view (FOV) of 780 mm. The transaxial FOV was 70 cm, and the matrix size was 128 × 128. Spatial resolution in air was 4.29 mm FWHM. The PET images were reconstructed from CT data for attenuation correction using the OSEM iterative algorithm with 20 subsets and two iterations. The Biograph mCT-64 PET/CT scanner acquired images with a slice thickness of 3 mm simultaneously for a longitudinal FOV of 500 mm. The transaxial FOV was 58.8 cm, and the matrix size was 256 × 256. Spatial resolution in air was 4 mm FWHM. The PET images were reconstructed from CT data for attenuation correction using the TrueX algorithm and an all-pass filter with 21 subsets and two iterations.max was measured using each dedicated PET workstation (ADW version 4.3 for Discovery STE-16 and syngo MI for Biograph mCT-64). The optimal SUVmax cut-off values were determined using receiver operating characteristic curve (ROC) analysis for the small and large LN groups. When the measured SUVmax exceeded the optimal cut-off value or 2.5, the LN was considered positive. In addition, subgroup analyses were performed according to the PET/CT scanner (PET A and B), T stage (early and advanced T stages), and F-18 FDG uptake of the primary tumor (low and high tumor SUVmax). Patients who were examined using the Discovery STE-16 PET/CT scanner were classified into the PET A group and patients who were examined using the Biograph mCT-64 PET/CT scanner were classified into the PET B group. Patients with T1 or T2 stage were classified into the early T stage group and patients with T3 or T4 stage were classified into the advanced T stage group. Patients with SUVmax of the primary tumor lower than 13.0, which was the median value of SUVmax, were classified into the low tumor SUVmax group and patients with SUVmax of the primary tumor higher than 13.0 were classified into the high tumor SUVmax group. The optimal SUVmax cut-off values were determined for each subgroup.An experienced nuclear physician blinded to the histopathologic and colonoscopic results reviewed the F-18 FDG PET/CT images on a workstation . The locations of the regional LNs were recorded as the perirectal, superior rectal, inferior mesenteric, or internal iliac areas. Suspicious lymph nodes less than 3 mm were ignored because they cannot be differentiated from vascular structures or other nonspecific soft tissue densities. The ROIs  were drawn in consensus around the regional LNs, and the SUVmax in each group and each subgroup were calculated using ROC analysis. The sensitivities, specificities, and accuracies of PET/CT using the optimal SUVmax cut-off values according to LN size, and a fixed SUVmax cut-off value of 2.5, were calculated for each group, each subgroup, and for all patients together. The areas under the curve (AUCs) of the optimal and fixed SUVmax cut-off values were compared using C-statistics. A p-value < 0.05 was considered significant.The optimal cut-off values of the SUVn = 118) or large (n = 58) LN groups. Regional LN metastasis was confirmed pathologically in 32 patients (27.1%) in the small LN group, and 35 patients (60.3%) in the large LN group.Of 296 patients who underwent preoperative F-18 FDG PET/CT and follow-up curative surgery for rectal cancer, 120 patients were excluded from this study according to the exclusion criteria . There was no significant difference in the SUVmax of small and large LNs between the PET A and B groups . The SUVmax of the small LN in the advanced T stage group was significantly higher than that in the early T stage group , but the SUVmax of large LN was not significantly different between these two groups . The SUVmax of the small LN in the high tumor SUVmax group was significantly higher than that in the low tumor SUVmax group , but the SUVmax of large LN was not significantly different between these two groups .The SUVmax for the diagnosis of regional LN metastasis were 1.1 in the small LN group, and 2.1 in the large LN group. The sensitivity, specificity, accuracy, and AUC using the optimal SUVmax cut-off values were 90.6, 70.9, 76.3%, and 0.808 in the small LN group, and 68.6, 78.3, 72.4%, and 0.734 in the large LN group, respectively . Figure max cut-off values, but not by the fixed SUVmax cut-off value of 2.5. There was no significant difference in the AUC between the two methods in the large LN group (p = 0.429).The optimal cut-off values of SUVp = 0.071).In overall patients, the sensitivity, specificity, accuracy, and AUC of F-18 FDG PET/CT, using the optimal cut-off values, were 76.1, 74.3, 75.0%, and 0.752, respectively whereas on using the fixed cut-off value, the sensitivity, specificity, accuracy, and AUC were 35.8, 97.2, 73.9%, and 0.665, respectively. The AUC of PET/CT using the optimal cut-off value was higher than that using the fixed cut-off value of 2.5 in all patients, but not statistically significant . There were no significant differences in the AUCs between PET/CT using the optimal and fixed cut-off values in the large LN of the PET A group (p = 0.866), as well as small and large LNs of the PET B group (p = 0.110 and p = 0.162). Subgroup analysis according to the T stage revealed that the optimal cut-off values were 0.9 for small LN and 1.8 for large LN in the early T stage group and 1.1 for small LN and 2.1 for large LN in the advanced T stage group. There were no significant differences in the AUCs between PET/CT using the optimal and fixed cut-off values in the small and large LNs of the early and advanced T stage groups . Additional file max of the primary tumor revealed that the optimal cut-off values were 1.0 for small LN and 1.3 for large LN in the low tumor SUVmax group, and 1.1 for small LN and 2.1 for large LN in the high tumor SUVmax group. There were no significant differences in the AUCs between PET/CT using the optimal and fixed cut-off values in the small and large LNs of the low and high tumor SUVmax groups . Additional file max groups.Subgroup analysis was performed according to the PET/CT scanner. In the PET A group, the optimal cut-off values of SUVmax cut-off values according to the size of the LN. Application of a lower SUVmax cut-off value to evaluate a small LN increased the sensitivity of PET/CT in the detection of metastatic LNs in patients with rectal cancer. The AUCs of the PET/CT with optimal SUVmax cut-off values were significantly higher than those with a fixed cut-off value of 2.5 in the small LN group. These results suggest that F-18 FDG PET/CT can diagnose LN metastasis as accurately in small LNs as in large LNs if a lower SUVmax cut-off value is applied. Although the concept of this hypothesis is widely known, the present study proved it practically in sufficient number of patients with rectal cancer.The present study revealed improved diagnostic performance of F-18 FDG PET/CT in the evaluation of metastatic LNs in patients with rectal cancer using the optimal SUVmax cut-off values according to the LN size allows determination of treatment strategies and improves the prognosis of patients with rectal cancer by improving the accuracy of the diagnosis of LN metastasis with F-18 FDG PET/CT.LN metastasis in rectal cancer is directly correlated with prognosis. The 5-year survival rate is > 95% in rectal cancer patients without LN metastasis, but decreases to 50~ 70% in patients with LN metastasis . Additiomax of 2.5 has been commonly used to diagnose metastatic LNs [max cut-off value of 2.5 in the present study were comparably low. However, there was significant improvement in the sensitivity of PET/CT when using an SUVmax cut-off value optimized according to LN size.F-18 FDG PET/CT is beneficial in the preoperative staging of rectal cancer, though it showed low sensitivity and accuracy in the diagnosis of LN metastasis , 13. Theatic LNs . Howevermax cut-off values were determined for the evaluation of small LNs than for the large LNs. Several techniques have been developed to calibrate the partial volume effect. In addition, many studies revealed significant improvement in the diagnostic performance of F-18 FDG PET/CT for the determination of small lesions after partial volume correction [max cut-off values according to LN size to compensate for the partial volume effect, which resulted in significant improvement in the AUC. This approach could be a more rapid and simpler method for calibration of the partial volume effect.The primary cause of the low sensitivity of F-18 FDG PET/CT in the diagnosis of LN metastasis is the partial volume effect –16, whicrrection , 18. Howmax cut-off value of 2.5 showed low sensitivity (35.8%) and high specificity (97.2%), whereas F-18 FDG PET/CT using SUVmax cut-off values optimized according to size showed high sensitivity (76.1%) and high specificity (74.3%). The diagnostic value of the present study is considered to be comparable to those of previous CT or MRI studies. However, direct comparison of diagnostic value is limited between the present study and previous CT or MRI studies, because there are heterogeneities between studies including differences in protocols, radiologists’ experience, approach to image interpretation, and methodologic quality. Further studies with comparison of diagnostic value between F-18 FDG PET/CT, CT, and MRI in the same patient population could provide important information in selecting diagnostic modalities for preoperative staging of rectal cancer.CT and MRI have conventionally been used to evaluate LN metastasis in rectal cancer by evaluating the size and shape of the LN , 6. A dimax and could have affected some of the results of the present study. However, previous studies have shown that the difference in the SUVmax of the same lesion between two different scanners is < 0.05 [max of small and large LNs between the two scanners. Furthermore, the difference between the optimal cut-off values of the two scanners was only 0.1 for small LNs. Although the effect of using two different scanners on the results of the present study would be negligible, further prospective studies involving the use of one PET/CT scanner and a large population are needed for more valid optimal cut-off values.The limitation of the present study was the use of two different scanners , which could not be avoided owing to the retrospective study design. The difference in the resolution and administered dose of F-18 FDG, according to the two different scanners, could have caused differences in the SUVs < 0.05 . In the max increases the sensitivity of F-18 FDG PET/CT for evaluation of the small regional LNs in patients with rectal cancer. F-18 FDG PET/CT using the optimized SUVmax cut-off values according to the LN size has the potential to show improved diagnostic performance for the detection of regional LN metastasis in patients with rectal cancer. Further prospective studies involving the use of one PET/CT scanner and a large population are needed.Application of the lower cut-off value of SUVAdditional file 1:Table S1. Comparison of diagnostic values between PET/CT using the optimized cut-off values and the fixed cut-off value of 2.5 in patients with early (T1–2) and advanced (T3–4) T stages. (DOCX 19 kb)Additional file 2:Table S2. Comparison of diagnostic values between PET/CT using the optimized cut-off values and the fixed cut-off value of 2.5 in patients with low (< 13.0) and high (> 13.0) SUVmax of primary tumor. (DOCX 17 kb)


PU.1 is an ETS-family transcription factor that plays a broad range of roles in hematopoiesis. A direct regulator of myeloid, dendritic-cell, and B cell functional programs, and a well-known antagonist of terminal erythroid cell differentiation, it is also expressed in the earliest stages of T-cell development of each cohort of intrathymic pro-T cells. Its expression in this context appears to give T-cell precursors initial, transient access to myeloid and dendritic cell developmental competence and therefore to represent a source of antagonism or delay of T-cell lineage commitment. However, it has remained uncertain until recently why T-cell development is also intensely dependent upon PU.1. Here, we review recent work that sheds light on the molecular biology of PU.1 action across the genome in pro-T cells and identifies the genes that depend on PU.1 for their correct regulation. This work indicates modes of chromatin engagement, pioneering, and cofactor recruitment (“coregulator theft”) by PU.1 as well as gene network interactions that not only affect specific target genes but also have system-wide regulatory consequences, amplifying the impact of PU.1 beyond its own direct binding targets. The genes directly regulated by PU.1 also suggest a far-reaching transformation of cell biology and signaling potential between the early stages of T-cell development when PU.1 is expressed and when it is silenced. These cell-biological functions can be important to distinguish fetal from adult T-cell development and have the potential to illuminate aspects of thymic function that have so far remained the most mysterious. Spi1 gene, is an ETS-family transcription factor with multiple roles in hematopoiesis. It is a lineage-specifying transcription factor that positively regulates many genes in the macrophage, granulocyte, dendritic-cell and B-cell lineages. Expressed at highest levels in monocytes/macrophages, at low or moderate levels in B cells, and transiently in early erythroid precursors, its action is also important or indispensable for sustained generation of all known hematopoietic precursors that have lymphoid developmental potentials  and DN3  stages. This expression timing relative to other developmentally regulated transcription factors is conserved between human and mouse  do not make myeloid cells under these conditions, and this difference between stages up to DN2 and stages from DN3 onward has been a highly consistent observation (in vivo conditions (Hes1 are expressed throughout the pro-T cell phases (ETP to DN3), until T-cell receptor (TCR) gene rearrangement  but not in T cells . This hyervation , 51–56. nditions ? A potennditions , 50, 58.nditions –61, and angement –64 [reviangement ]. Thus, a priori, other than proliferation. In the past 5 years, however, a detailed look at the molecular biology of PU.1 action on the genome in pro-T cells has revealed much about the ways that PU.1 works, the complex cell biology of the early precursor states, and previously under-appreciated principles of transcription factor systems operating in development.The question raised by such results, however, was why PU.1 should continue to be expressed at all by cells once they entered the thymus. Population dynamic models imply that the stages when PU.1 is expressed occupy a minimum of 7–10 intrathymic cell divisions of pro-T cells , 66, 67.Spi1 knockout) mice with unconditional, germline mutations  are powerful because of the easy accessibility of the developing cells during differentiation and because of the ability to follow differentiation of a synchronized cohort of cells in absolute time. As described below, however, the genes most sensitively regulated by PU.1 in developing T cells suggest that this factor may be important to endow cells with additional functions as well, functions that may only contribute to their development specifically in the thymus in vivo.The Spi1fl/fl strain mice (Spi1) can target rapid, biallelic disruption of the Spi1 locus , the transduced cells as a whole reactivate little of the program that forms the normal context for endogenous PU.1 activity in pro-T cells  or OP9-DLL4 (OP9-DL4) stroma with IL-7 and Flt3L  of retrograde differentiation. Both Scid.adh.2C2 and normal DN2b/DN3 pro-T cells make strong responses to forced expression of PU.1, as noted above, and often the response includes downregulation of multiple later T-cell differentiation genes. Does this shed light on PU.1's natural role in earlier T-cell development, or is it simply an inhibitory artifact of overexpression? Clues that the gain-of-function phenotype is linked with a genuine role of PU.1 in earlier T-cell development come from PU.1's (re-)activation of a group of genes that are specifically associated with the early progenitor state, including and Lmo2 , 79, 85.and Lmo2 .Notch1 itself. Whether cause or effect, this collapses the balance between Notch signaling and PU.1 activity that is fundamental to channel the natural role of PU.1 in early pro-T cells (see above)  , 58. Thee above) , and thie above) , 58. Impe above) , 50, 58.Il2ra), and often upregulate the myeloid-associated marker CD11b . The gene expression profiles of cells losing CD25 and upregulating CD11b are radically transformed from the state of newly-committed pro-T cells within 2 days after transduction, with widespread repression of T-lineage-affiliated transcription factor genes and Notch target genes as well as upregulation of multiple Cebp and Irf family transcription factor genes . Th. ThIl2raor genes , 85. DetSpi1 allele (Spi1fl.fl) is asynchronous, and in an early T-cell population with mixed degrees of Spi1 deletion, cells with inadequate PU.1 levels appear to be at a selective disadvantage, even in vitro. Ironically, because PU.1 protein can persist longer than a cell cycle (Spi1 (see above) can also interfere with the dilution needed to complete the clearance of the PU.1 protein. As a result of the enrichment of cells with undeleted alleles, and this persistence of pre-existing PU.1 protein even from the cells that have successfully deleted its coding gene, the effects on target gene regulation appear very weak at timepoints up to 2 days after PU.1 deletion, despite the fact that the reduced cell yields from the knockout cells show that PU.1 is biologically important , to compete for binding against endogenous PU.1   , 93. In 1b gene) . Althoug1b gene) .The sites where PU.1 binds are enriched for open chromatin as defined by DNase accessibility or ATAC-seq [assay of transposase-accessible chromatin ], and ciBecause PU.1 mediates different effects in the rather different regulatory contexts of B, dendritic, myeloid, erythroid progenitors and pro-T cells, an important question is how much of PU.1's binding choice hierarchy is dependent on the prior epigenetic history of cells. PU.1 cannot enter all genomic sites. Notably, PU.1 appears to be excluded from genomic regions that are packaged in Polycomb Repressive Complex 2-modified chromatin, as marked by trimethylation of Histone H3 Lysine 27 (H3K27me3) . HoweverStill to be determined are the rules determining when PU.1 binding to closed chromatin results in opening of the closed site. A priori, one could imagine that PU.1 establishes occupancy using its DNA-binding domain and then uses its protein-interaction domains to recruit chromatin modifying complexes ]. Howevechange in PU.1 binding at a given site with the rapid, measurable change in expression of the target gene linked to that site. This is considerably easier to do in a gain of function format than in loss of function, as an epitope-tagged exogenous PU.1 construct can be introduced with fast kinetics and its newly established binding tested for association with local gene expression responses (+ CD11b− (see above) were used for ChIP-seq analysis of exogenous PU.1 binding.The major problem with connecting PU.1 binding to PU.1 regulatory function is that PU.1 binds to too many genomic sites in ETP and DN2a pro-T cells . It showesponses . Note thesponses  only celThe results showed that PU.1 exerts its main functional regulatory impacts in pro-T cells via non-promoter sites, and especially via sites that are normally developmentally changing in chromatin accessibility  Figure . In the The impact of PU.1 on pro-T cell gene expression overall is at odds with the biochemical and genomic evidence for its mode of action in one respect: PU.1 introduced into primary pro-T cells or Scid.adh.2C2 cells causes downregulation of many T-cell genes, especially those associated with Notch signaling and TCR gene rearrangement after commitment. This response is fast, reducing existing transcript pools for many repressed genes even before most positively regulated PU.1 target genes are seen to be turned on . HoweverThere is a long history of research on PU.1 as a repressor of genes associated with non-myeloid pathways, especially in the context of PU.1—GATA-1 antagonism in hematopoiesis .Notch1 itself  not only silence ry genes . They alry genes , but has complex . AlthougC2 cells , when ac  in human pro-T cells (Thy1—Thy4) are similar to their patterns in murine pro-T cells , Ets1, and Lef1 are strongly affected in cells making a lineage switch, but none of these are measurably repressed in cells within the T-cell path. The cell cycle-regulatory locus E2f2, which is also upregulated during commitment, is rare among transcription factor coding genes in that it does appear to be under active repression by PU.1 until the transition to commitment. Thus, within the T-cell pathway, PU.1 has a specific role in promoting maintenance of certain Phase 1 regulatory genes before commitment, but little role in repressing T-cell differentiation regulators directly.Data from multiple studies show that PU.1 regulates a subset of developmental control genes but is not alone in its actions. Varied PU.1 gain and loss of function perturbations in the DN2-DN3 stages show that PU.1 does provide positive input into a discrete subset of Phase 1 regulatory genes, with nt tests , 80, 85.-T cells . PU.1 is-T cells , 123. Addeclines , 124  as well as cytoskeletal components such as Coro2a and Myo1f. Representative samples of the expression patterns of such genes are shown in Potentially important clues to PU.1 roles are the prominence among positively regulated PU.1 targets of genes encoding specific cytokine receptors not yet studied in T-cell biology ; multiple protein tyrosine kinases ; and G-protein coupled receptors  and G protein signaling mediators (Gng2 and Rgs18); while PU.1 represses other G protein signaling mediators (Gimap and Gbp family members). In addition, PU.1 directly promotes expression of cell surface molecules  used as markers for stages in early T-cell development, but which in vivo and in vitro   . Thus, pyolk sac , 133. Ityolk sac  and in t systems . This is systems  and due  systems –139.in vivo or in vitro, with functional and phenotypic defects evident in the mutants in both multipotent progenitors and newly-entered intrathymic ETPs, as early as in the late fetus. This difference in PU.1 dependence accompanies a subtly different T-cell developmental program. Gene expression differences have been noted between the normal first-wave fetal and adult pro-T cells in the thymus at corresponding stages which indicate that the fetal program drives accelerated development (Remarkable differences have been reported between genetic requirements for T cell development derived from earlier and later waves of prethymic progenitors. For example, the crucial T-lineage transcription factor TCF-1, which plays roles in numerous phases of thymocyte development , 140–145elopment , 149. Moelopment . Thus, nThe first-wave precursor cells, also uniquely, enter the thymus by a different route than all subsequent waves. Instead of entering through the blood vessels near the cortical-medullary junction, these early cells migrate through cervical-region mesenchyme to the thymic anlage before it is vascularized. The thymus does not yet have a capsule to present a physical barrier, and the first-wave cells enter directly through the future outer cortex. Thus, they may not use the same interactions with basement membrane, endothelial cells, or chemokine gradients as any future wave of thymic precursors. Not only are these cells intrinsically programmed to cut short the stages supported by PU.1-dependent transcriptional regulators, but also they can dispense with many of the cell biological tools that PU.1 may provide to later-wave successors to navigate the adult or late-fetal thymus.PU.1 is a broad regulator of the properties of the cells that first enter the thymus, and it helps to determine their proliferation and rate of progression to commitment after they arrive. While dysregulated PU.1 can cause trans-differentiation to myeloid or dendritic-cell fates, endogenous PU.1 normally plays a protracted role within early T-cell development. Its target genes are occasionally repressed but mostly activated by PU.1 binding, and they confer on the cells distinctive stage-specific transcription factor expression patterns as well as a rich array of stage-specific cell biological features that await proper functional analysis. This positive regulatory role is one result of PU.1's strong pattern of binding across the genome, its prominent occupancy of open chromatin sites, and the evidence that it helps to maintain the open chromatin states at bound regulatory sites as long as it is expressed. The number of genes that respond quickly to changes in PU.1 activity may only account for a minority of all the genomic sites where PU.1 is found engaged; at other sites, its role could be structural or redundant with other factors. However, it is clear that PU.1 also affects the activity of certain genes that it does not bind to directly, via creating preferential interaction sites for other factors that can deplete the regulatory elements of those factors' alternative target genes. Through chromatin state placeholding and “coregulator theft” as well as through its own direct transcriptional activities, PU.1 pervades the regulatory state of early T cells as long as it is expressed.per se.This phase comes to an end when other transcription factors finally accumulate to the point where they can shut PU.1 off. The best current candidates for this silencing activity include GATA-3 , 105, TCTcf7 and Gata3 until they reach the thymus. Is PU.1 part of the answer? The system-wide impact of PU.1 on other factors suggests that in scenarios where PU.1 is absent, the same T-cell transcription factors might initially choose different binding sites. Indeed, pro-T cells that have PU.1 acutely deleted at an early stage do not only differentiate faster along the T lineage; they also tend to shift to a natural killer-like program more readily than controls (The pioneering role of PU.1 on the genome raises fascinating questions for future study that connect mechanism with developmental lineage selection. Hematopoietic progenitors express PU.1 before they enter the thymus, but the pattern of its occupancy is not well defined at that stage, so the onset of PU.1's pioneering activity in precursors that will eventually generate T cells is not easy to study. The mechanisms discussed in this review show that it establishes a pre-pattern that can influence the binding of the other transcription factors expressed in the cell throughout multiple cell cycles in the thymus. It is not clear, though, how this particular pre-pattern is set, to be distinguished from PU.1 binding patterns in B cells and myeloid cells . The quecontrols . Thus, aER wrote the paper, contributed to ideas in the review, and directed research that led to this review. HH and JU carried out research that led to this review, contributed to ideas in the review, provided some figures and edited the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Annona cherimola Mill is a large green fruit with black seeds widely known to possess toxic properties due to the presence of Annonaceous acetogenins. The present study investigates the anti-cancer properties of an Annona cherimola Mill ethanolic seed extract on Acute Myeloid Leukemia (AML) cell lines in vitro and elucidates the underlying cellular mechanism. The anti-proliferative effects of the extract on various AML cell lines and normal mesenchymal cells (MSCs) were assessed using WST-1 viability reagent. The pro-apoptotic effect of the extract was evaluated using Annexin V/PI staining and Cell Death ELISA. The underlying mechanism was deciphered by analyzing the expression of various proteins using western blots. Treatment with an A. cherimola seed ethanolic extract promotes a dose- and time-dependent inhibition of the proliferation of various AML cell lines, but not MSCs. Positive Annexin V staining, as well as DNA fragmentation, confirm an increase in apoptotic cell death by upregulating the expression of pro-apoptotic proteins which control both intrinsic and extrinsic pathways of apoptosis. GC/MS analysis revealed the presence of phytosterols, in addition to other bioactive compounds. In conclusion, Annona cherimola Mill seed extract, previously known to possess a potent toxic activity, induces apoptosis in AML cell lines by the activation of both the extrinsic and the intrinsic pathways. Annona cherimola Mill. and member of the custard apple family (Annonaceae), is a large green fruit with round protrusions, white pulpy flesh, and black bean-size seeds embedded in the pulp. Annona cherimola Mill. is the official name of the plant, as featured on theplantlist.org [Cherimoya, fruit of the tree of list.org .A. cherimola extract has been used for various purposes, mostly due to its potent toxic activity [A. cherimola seed extract with the eyes incidentally caused blindness and ingestion of the extract caused gastrointestinal disturbances such as nausea, vomiting, flatulence, and atropine-like effects, including photophobia and dryness of the mouth [Traditionally, activity . Crushedactivity . In facthe mouth .A. cherimola [Important toxic components of the seeds include the annonaceous acetogenins (ACGs). These are considered environmental neurotoxins responsible for neurodegenerative diseases like atypical Parkinsonism and dementia in areas known for cultivating herimola . Moreoveherimola .A. squamosa and A. muricata seed extracts have been used as natural insecticides against Madagascan mosquitoes [A. muricata [A. muricata were shown to be neurotoxic against dopaminergic neurons, which explains the neuronal dysfunction and degeneration underlying the West Indian parkinsonian syndrome [Furthermore, close species of the Annona genus have also been shown to possess toxic activity. In fact, squitoes . Additiomuricata . Some ofmuricata . Other psyndrome . All theA. cherimola is considered to have some health benefits when included in the diet [On the other hand, the diet . Extractthe diet . It has the diet .A. cherimola seeds exhibited cytotoxic effects against prostate, breast, and colon cancer cell lines, with a 10,000 times stronger potency than adriamycin, an important chemotherapeutic drug [Recently, research has investigated the pharmacological activities exhibited by different parts of the plant, notably leaf extracts, which showed cytotoxicity against several microbial species, as well as breast, colon, and liver cancer cell lines . Annonactic drug . HoweverAcute Myeloid Leukemia (AML) is a cancer that affects the blood and bone marrow and it includes a group of leukemia that develops in all blood cells, excluding lymphocytes . TreatmeA. cherimola on AML cell lines in vitro and elucidates its mechanism of action.The present study investigates the anti-cancer properties of the known toxic extract from the seed of A. cherimola seed ethanolic extract (ASEE), was calculated and the results show a dose- and time-dependent decrease in the proliferation of AML cell lines used. In fact, a significant time-dependent decrease in the proliferation of KG-1 cells is observed at all concentrations, with an IC50 of 57 µg/mL at 24 h and 20 µg/mL at 48 h , treated with  at 48 h A. A simi at 48 h B. In Mon at 48 h C. On the at 48 h D. Moreov < 0.001 .Using Propidium Iodide (PI) to assess the DNA content of cells, the distribution of the cells in the different cell cycle stages is elucidated and it shows a shift of the cells from the G0-G1, S, and G2-M stages to the sub-G1 stage, indicating cellular fragmentation. The distribution of KG-1 cells after 24 h follows a dose-dependent change between the control group, where only 2.8% of cells are in the sub-G1 stage, 56.8% in the G0-G1 stage, 17.4% in the S stage, and 16.5% in the G2-M stage, compared to the cells treated with 114 µg/mL, where 59.8% of cells are in the sub-G1 stage, 13.1% in the G0-G1 stage, 11% in the S stage, and 0.9% in the G2-M stage . The same dose-dependent shift in the distribution of cells is observed in Monomac-1, whereby the percentage of cells in the sub-G1 stage increases from 11.5% in the controls to 63.7% upon treatment with 152 µg/mL ASEE for 24 h .Using fluorescence microscopy, Annexin V binding to the cell membrane is detected, which is concomitant with the occurrence of apoptosis that is accompanied by flipping of the phosphatidyl serine moieties to the outer leaflet of the cell membrane. To quantify the increase in apoptosis, Annexin V/PI dual staining was performed, followed by flow cytometry and analysis. Normal living cells exhibit negative staining to both PI and Annexin (lower left quadrant). However, early apoptotic cells exhibit negative staining to PI, but positive staining to Annexin (lower right quadrant), and late apoptotic cells exhibit positive staining to PI and Annexin (upper right quadrant). Necrotic cells exhibit positive staining to PI and negative staining to Annexin. In KG1 cells, we observe an increase in apoptosis upon treatment with 114 µg/mL of ASEE for 24 h, whereby 31.8% are in early apoptosis and 62.8% in late apoptosis, compared to 5.8% and 3.1% in untreated cells, respectively . A similTo further confirm the increase in apoptosis, Cell Death ELISA was performed to quantify the levels of DNA fragmentation, which is a major hallmark of apoptosis. A significant dose-dependent increase in the enrichment factor is noted for both AML cell lines after 24 h. A 1.6-fold increase and 2.5-fold increase in the enrichment factor are observed in KG-1 cells treated with 38 µg/mL and 76 µg/mL, respectively (before and after the IC50 = 57 µg/mL) A. A simiTo identify the pathway by which ASEE inhibits proliferation and induces apoptosis, protein extraction from KG-1 cells was treated for 24 h with different concentrations of ASEE before and after the IC50 was performed, followed by SDS-PAGE and western blot for the quantification of key cell proliferation and apoptosis-regulating proteins. The results show an upregulation of key pro-apoptotic proteins such as p53, as well as cleaved PARP-1. The upregulation in Cytochrome-c indicates its release from the mitochondria. An increase in the agents of apoptosis, such as cleaved caspase-8 and cleaved caspase-9, is also detected, and an increase in the Bax to Bcl-2 ratio is noted as well .Using the 2,7-dichlorofluorescin diacetate (DCFDA) Cellular Reactive Oxygen Species (ROS) Detection Assay kit, the ROS levels were measured, depending on the cleavage of the reduced non-fluorescent form of fluorescein to its fluorescent form by agents of oxidation in KG-1 cells. In fact, no significant change is observed in the ROS levels in the cells treated with ASEE alone, which indicates that the extract alone does not significantly induce or change the level of ROS in living cells. However, the addition of Tert-Butyl Hydrogen Peroxide (TBHP) (75 μM), which is known to be an ROS inducer, significantly increases the ROS level up to a 5.25-fold increase. Furthermore, adding 75μM of TBHP along with increasing concentrations of ASEE results in a gradual decrease in the ROS level induction by TBHP from a 5.25-fold increase in 75 μM TBHP only, down to a 2.98-fold increase in 75 μM TBHP with 114 µg/mL, which is a significant decrease when compared to the TBHP-only treated cells. This clearly indicates that ASEE has antioxidant properties since it can quench ROS induction by TBHP .To explore the composition of the extract, ASEE was analyzed using Gas Chromatography-Mass Spectrometry (GC-MS) in order to detect the presence of any small molecular weight, nonpolar, and volatile compounds possibly responsible for the anticancer properties of the extract. Several compounds were detected and most of them were identified ; Table 1A. cherimola seeds are considered an important source of plant toxins which have been used as insecticides and linked to neurodegenerative diseases [A. cherimola Mill fruit was prepared due to the mild polarity index of ethanol [50 levels of 57 µg/mL, 107 µg/mL, and 100 µg/mL, respectively. These concentrations are potentially therapeutically effective considering that a previous study showed that the ethanolic extract from Urtica membranacea possessed potent anti-cancer effects at 750 μg/mL and 1500 μg/mL and these concentrations were efficiently correlated to mice breast cancer model treatment with no side effects [diseases ,4. Howevdiseases . For tha ethanol , allowin ethanol . The res effects .The results also revealed a mild cytotoxic effect of the extract on MSCs, which was statistically lower than the cytotoxicity observed in AML cell lines. This further predicts the efficacy of the extract in targeting cancerous cells with minimal effects on normal cells, which is a crucial advantage of a chemotherapeutic drug with target selectivity . The cytTo explore the mechanism by which this previously considered toxic extract is exerting its anti-proliferative effects on the AML cell lines, KG-1 and Monomac-1 cells, which were more responsive to ASEE, were evaluated for cell cycle arrest and for any apoptotic changes. All experiments showed that the extract induced a dose-dependent increase in apoptosis levels in the cells, supported by an increase in cellular fragmentation, a flipping of the phosphatidylserine moieties to the outer leaflet of the cell membrane along with an increase in Annexin V staining of the cells, and an increase in DNA fragmentation. These apoptotic hallmarks, increasing between the control cells and the cells treated with the extract, are the result of the cellular activation of apoptotic pathways, by the mediation of several proteins . MovemenInvestigation of the apoptotic mechanisms activated in the cells treated with ASEE showed that both the intrinsic and extrinsic pathway of apoptosis are activated in a dose-dependent manner upon treatment and they coalesce to activate the execution pathway of apoptosis. The execution pathway is the final phase of apoptosis signaling where various intracellular mediators are activated, leading to the biochemical and morphological changes observed in apoptosis . Among tAs mentioned previously, ASEE treatment of AML cell lines also promoted apoptosis via an intrinsic pathway. This pathway is usually induced by stimuli that affect targets within the cell and cause mitochondrial-initiated events. Loss of mitochondrial transmembrane potential is usually followed by cytochrome-c release into the cytosol. Cytochrome-c then binds and activates pro-caspase-9, which leads to the upregulation of cleaved caspase-9. Other groups of proteins are released from the mitochondria and they are responsible for the DNA changes observed in apoptosis and detected by Cell Death ELISA . This isOther proteins in the intrinsic pathway are effectors of p53-mediated apoptosis. The significant increase in p53 levels observed can be correlated to the upregulation of the apoptotic machinery by increasing the release of cytochrome-c from the mitochondria, and it can also lead to cell cycle arrest related to growth retardation. p53 is also speculated to regulate some members of the Bcl-2 family; however, the mechanism of inhibition is still not well-elucidated .Moreover, ASEE also mediated apoptosis via the extrinsic pathway. The extrinsic or death receptor mediated pathway typically leads to the activation of caspase-8; our results revealed an upregulation of caspase-8, which was detected upon treatment with 76 µg/mL of ASEE. This suggests that the extrinsic pathway is only activated at high concentrations and that the intrinsic p53-dependent pathway plays a more crucial role in promoting the cell death of AML cells upon ASEE treatment.Although ROS are known to activate apoptosis in the cell by causing the damage of subcellular components , antioxiThe analysis of the composition of the extract revealed the presence of β-sitosterol. β-sitosterol is a phytosterol previously studied for its anti-cancer effects; it was shown to have anti-proliferative and pro-apoptotic effects on U937 cells by selectively activating caspase-3 and increasing the Bax/Bcl-2 ratio, whereby the addition of z-DEVD-fmk (a caspase-3 specific inhibitor) or the overexpression of Bcl-2 significantly attenuated the apoptotic response . Its abiAnother detected compound was β-stigmasterol, which is a plant derivative of cholesterol. Even though it is less studied than β-sitosterol, it was shown to inhibit some cancer cells in vitro, like human laryngeal carcinoma cells . As for A. cherimola seeds [Finally, acetogenins which have previously been reported in la seeds  could noAnnona cherimola Mill., a traditionally used plant seed with toxic properties, has been shown to exhibit a promising cytotoxic effect on AML cell lines, by inducing apoptosis through a p53-dependent mechanism and through the induction of both the intrinsic and extrinsic apoptotic pathways and decreasing the ROS levels.In conclusion, the ethanolic extract from the seeds of TM, Dublin, Ireland) and antibiotics  in a humidified incubator in 5% CO2 at 37 °C. Before plating, cell viability was checked using the ZOE Fluorescent Cell Imager , along with Trypan Blue exclusion method.The Acute Myeloid Leukemia cell lines Mono-Mac-1, U937, and KG-1 were obtained from the American Type Culture Collection. The cells were cultured in Roswell Park Memorial Institute medium  enriched with 10% fetal bovine serum  supplemented with 100 U/mL penicillin and 100 μg/mL streptomycin (Lonza). After removing the bone epiphyses with sterile scissors, bone marrows were flushed out using a needle filled with Dulbecco’s Modified Eagle Medium  supplemented with 10% Fetal Bovine Serum (GibcoTM) and 100 U/mL penicillin and 100 μg/mL streptomycin (Lonza). The cells collected were then incubated in vented flasks at 37 °C with 5% CO2. After 5 days of daily medium change, MSCs were identified by their spindle-shaped morphology, as observed using the ZOE Fluorescent Cell Imager [MSCs were isolated from rat bone marrow according to a modified procedure. The single, 12-weeks old rat was provided by the animal facility at the Lebanese American University. It was maintained under optimal laboratory conditions and received food and water ad libidum, complying with the University’s Animal Care and Use Comity (ACUC) and the Guide for the Care and Use of Laboratory Animals ,41. The l Imager ,43.Annona cherimola Mill fruits were taken from a tree in Awkar-Lebanon , during January 2018 and were identified by the botanist Dr. Nisrine Machaka-Houri, according to the indications and characteristics described by Vanhove (2008) [e (2008) . A vouchFruit seeds were grinded and shaken with 80% ethanol at 200 rpm for 1 week. The extract was then filtered through a cheesecloth and spun at maximum speed to discard the pellet. Ethanol was evaporated using a rotary evaporator. The dried extract was weighed and then dissolved in Dimethyl sulfoxide (DMSO) and diluted with RPMI to obtain a crude extract with a final concentration of 3800 µg/mL. When applied on the cell lines, the DMSO level maximally reached 0.4% at 150 µg/mL. 5 cells/well and incubated overnight. Triplicates of wells were treated with increasing concentrations of A. cherimola seeds ethanolic extract (ASEE) (9.5–152 μg/mL). Control cells were treated with RPMI media. The plates were incubated for 24 h or 48 h before the addition of WST-1 cell viability reagent , according to the manufacturer’s guidelines. Absorbance at 450 nm was measured by spectrophotometry using a MultiskanTM FC Microplate Photometer to detect metabolically active cells and the percentage proliferation was calculated.AML cells and MSCs were seeded in 96-well plates at a density of 0.5 × 105 cells/well and incubated overnight. After incubation for 24 h or 48 h with increasing concentrations of the ASEE , the cells were fixed with ice-cold absolute ethanol for fixation and stained with PI . The DNA content was assessed using the Accuri C6 flow cytometer. The distribution of cells in each cell cycle phase was determined by assessing the DNA content: sub-G0/G1 phase cells (Pre-G or dead cells) have <2n, G0/G1 phase cells have 2n, S phase cells have between 2n and 4n, and G2/M phase cells have 4n.AML cells were seeded in 6-well plates at a density of 1 × 105 cells/well and incubated overnight. After incubation for 24 h or 48 h with increasing concentrations of the ASEE , cells were stained with Annexin using the Annexin V–Fluorescein Isothiocyanate (FITC) Apoptosis Detection Kit . The cells were visualized under the ZOE Fluorescent Cell Imager using bright-field conditions and the filter set was then for FITC before merging the images.AML cells were seeded in 24-well plates at a density of 1 × 105 cells/well and incubated overnight. After incubation for 24 h or 48 h with increasing concentrations of the ASEE , cells were stained with Annexin V and PI  and immediately analyzed using the Accuri C6 flow cytometer. Annexin V binds in a Ca2+-dependent manner to the exposed charged head groups of phosphatidylserine, which is translocated to the outer leaflet of the cell membrane upon apoptosis. The cell membrane integrity excludes PI in viable and apoptotic cells, but not in necrotic cells. Therefore, dual parameter FACS (Fluorescence-activated cell sorting) analysis allows for the discrimination between viable, apoptotic, and necrotic cells.AML cells were seeded in a 6-well plate at a density of 2 × 105 cells/well and incubated overnight. Duplicates of wells treated with two increasing concentrations of ASEE (38–76 μg/mL for KG-1 and 76–152 μg/mL for Monomac-1) were plated for 24 h. Control cells were treated with RPMI media. Positive control wells of cells were treated with 100 μM (58.85 µg/mL) of etoposide . Using the Cell Death ELISA kit , cells were extracted and lysed with incubation buffer before the isolation of fragmented cytosolic DNA. Microplate wells were coated with anti-histone antibodies. Extracted DNA was then incubated in the wells, which were washed before adding anti-DNA antibodies linked to an enzyme, and then washed again before adding an ABTS  colorimetric substrate. Absorbance at 405 nm (and 492 nm as a background) was measured by spectrophotometry using the MultiskanTM FC Microplate Photometer and DNA fragmentation enrichment factor (absorbance of treated cells/absorbance of non-treated cells) was calculated based on manufacturers’ instructions.AML cells were seeded in a 12-well plate at a density of 0.25 × 106 cells/mL before treatment with two increasing concentrations of ASEE for 24 h (38 and 76 μg/mL). Control cells were treated with RPMI media. Total proteins were extracted using the Q-proteome Mammalian Protein kit  and quantified using the DC (Detergent Compatible) protein assay (Bio-Rad). Proteins were separated by SDS-PAGE; transferred to PVDF (Polyvinylidene fluoride) membranes which were blocked with 5% skimmed milk; and then incubated with primary antibodies anti-β-actin , anti-Cytochrome-c, anti-cleaved PARP-1, anti-Bax, anti-Bcl2, and anti-caspase-9 , anti-p53, and anti-caspase-8  at the manufacturer’s recommended concentrations. After washing and incubation with a secondary antibody , membranes were washed and image development was done using the Clarity™ Western ECL Substrate  on the ChemiDoc machine . Blot bands were quantified using the ImageJ computer program to calculate the relative expression of proteins.KG-1 cells were plated in a 6-well plate at a density of 102DCFDA), and then plated in duplicates in a 96-well plate and treated with either increasing concentrations of ASEE (19–114 μg/mL), or with a combination of 75 μM of Tert-Butyl Hydrogen Peroxide (TBHP) with increasing concentrations of the ASEE (control cells were treated with RPMI media). TBHP is a potent ROS inducer inside the cells, so it was used as a positive control. H2DCFDA  acts as an indicator for ROS in cells upon its oxidative conversion to the highly fluorescent 2′,7′-dichlorofluorescein (DCF), which was quantified by fluorescent spectroscopy on the Varioskan™ LUX multimode microplate reader .The DCFDA Cellular ROS Detection Assay kit  was used to detect the levels of Reactive Oxygen Species (ROS) in the cells. KG-1 cells were incubated with the cell-permeant 2′,7′-dichlorodihydrofluorescein diacetate . The percentage composition was computed from GC peak areas.p-values were calculated by t-tests or two-way ANOVA, depending on the experiment. Significant differences were reported, with * indicating a p-value: 0.01 < p < 0.05, ** indicating a p-value: 0.001 < p < 0.01, *** indicating a p-value: 0.0001 < p < 0.001, and **** indicating a p-value: p < 0.0001.All the experiments were repeated three times. Statistical analyses were performed using GraphPad Prism 8 . The error bars were given as the mean ± SEM and the


Using pMHC multimer staining and sorting of NW8-specific T cells, the TCR repertoire raised against NW8 was characterised here using high-throughput sequencing in 20 HLA-B*44:03 subjects. We found that the CD8+ T cell repertoire raised in response to NW8 was highly skewed and featured preferential use of a restricted set of V and J gene segments. Furthermore, as often seen in immunity against ancient viruses like CMV and Epstein-Barr virus (EBV), the response was strongly dominated by identical TCR sequences shared by multiple individuals, or “public” TCRs. Finally, we describe a pair “superdominant” TCR clonotypes, which were germline or nearly germline-encoded and produced at remarkably high frequencies in certain individuals, with a single CMV-specific clonotype representing up to 17% of all CD8+ T cells. Given the magnitude of the NW8 response, we propose that this major skewing of CMV-specific immunity leads to massive perturbations in the overall TCR repertoire in HLA-B*44:03 individuals.Lack of disease during chronic human cytomegalovirus (CMV) infection depends on the maintenance of a high-frequency CMV-specific T cell response. The composition of the T cell receptor (TCR) repertoire underlying this response remains poorly characterised, especially within African populations in which CMV is endemic from infancy. Here we focus on the immunodominant CD8+ T cell response to the immediate-early 2 (IE-2)-derived epitope NEGVKAAW (NW8) restricted by HLA-B Human cytomegalovirus (CMV) is a highly prevalent β-herpesvirus , 2. AlthWhether the size of the CMV-specific T cell response impacts the ability of the immune system to mount effective responses against other pathogens is unclear, but it is apparent that HIV disease progression is substantially more rapid in the case of HIV-CMV coinfection , 15. Age*44:03  is specific for the immediate-early-2 (IE-2463−473)-derived epitope NEGVKAAW (hereafter referred to as NW8). The disproportionate magnitude of the NW8-specific specific response in B*44:03+ individuals is likely to be underpinned by strong biases in the TCR repertoire, leading to preferential selection of NW8-specific CD8+ T cells against this HLA background, although this possibility has yet to be investigated.We recently characterised an immunodominant CMV-specific response in African populations that is restricted by HLA-B*44:03 . This cl*44:03  and diff , or near germline (β chains). We conclude that the high-magnitude NW8 response causes a major perturbation of the TCR repertoire, with disproportionate expansion of germline clonotypes that are highly shared amongst HLA-B*44:03+ individuals.Using high-throughput sequencing, we sought to characterise the TCR repertoire of peptide-MHC tetramer-sorted NW8-specific CD8+ T cells from 20 HLA-B3  and a median HIV viral load of 20,500 copies/ml . HIV viral load was measured using the Roche Amplicor version 1.5 assay according the manufacturer's instructions; CD4+ T cell counts were measured by flow cytometry. Samples utilised for global TCR repertoire sequencing from genomic DNA were obtained from subjects of similar ethnic background and also recruited in Durban, South Africa. Ethics approval, for both studies, was given by the KwaZulu-Natal Review Board (Durban cohort). All subjects provided written informed consent.We studied 20 HIV-infected ART-naive adult subjects from a previously described cohort in Durban (South Africa) . The subPeptide-MHC tetramers were generated as previously described . CryopreRNA extraction was carried out using the RNEasy Micro kit  as previously described . BrieflyTCR sequencing from genomic samples from whole blood was carried out by Adaptive Biotechnologies, using the Immunoseq platform,as previously described .Analysis of the raw TCR sequences was performed using MiXCR . MiXCR eJ′):TCR repertoire clonality is given by the Shannon evenness index :Compositional similarity between TCR repertoires were assessed using the Morisita-Horn similarity index  in both samples. The similarity indices range in value from 0  to 1 . The CMH similarity index accounts for both the number of common clonotypes and the distribution of clone sizes and is sensitive to the clone sizes of the dominant clonotypes. CMH similarity calculations were performed using the “numpy” package in Python.where P values < 0.05 were considered significant.All pairwise statistical tests were performed in Prism v7.0  unless stated otherwise. Strength of association between two variables was analysed by Spearman's rank test. *44:03+ individuals from Durban, South Africa. The mean size of the tetramer+ population was 3.88% of CD3+CD8+ T cells (IQR 0.36-6.05) and the mean number of cells sorted for sequencing was 10,169 cells   and J gene segments . AntigenMH) to perform pairwise comparisons between every combination of two donors, as a measure of compositional similarity, or overlap  and 1 . The CMH value for every combination of two donors, representing a total of 120 pairwise combinations for the TCR-α chain and 153 for TCR-β . The mean CMH value of the TCR-α chain repertoire was 0.20 ± 0.02 . These clones were also the most extensively shared. CAVGNNAGNMLTF was found in 12/16 (75%) of subjects studied and CAVGANAGNMLTF in 7/16 (44%). To confirm this extreme bias, TCR-α chains were sequenced using the Immunoseq platform from whole blood genomic DNA of 26 individuals of the  same ethr = 0.4290, p = 0.0015; Figure *44:03+ individuals.Similarly, the most abundant public TCR-β clonotypes, CASSIFGEQFF and CASSIFGELFF , were shared by 7/18 (39%) of individuals studied. Consistent with previous observations , the higr = 0.798, p < 0.0001, Supplementary Figure The public amino acid sequences were often encoded by more than one distinct nucleotide sequence  or with the GGS motif  composed of a single TCR, CAVGANAGMLTF-CASSIFGEQFF. This, to our knowledge, is the largest reported T cell response mounted by a single public T cell clone in humans, which initially led us to envisage a positive relationship between TCR publicity and the magnitude of the T cell response. However, we found that this was not the case, as the number of public TCRs did not correlate with the percentage of tetramer-positive cells or with the number of sorted cells. Instead, the frequency of a given clonotype positively correlated with the extent to which it is shared . Altogether, these results suggest that publicity in T cell responses does not necessarily equate to superior immunity. Rather, the dominance of public TCRs within an individual and across individuals can be explained by their high precursor frequency, or in other words, by a numerical competitive advantage .The patient cohort recruited for this study were HIV-1 co-infected and receiving ART and it is well-established that such individuals have heightened CMV-specific T cell responses. Whether the expansion of superdominant clonotypes stems from dysregulated and heightened responses resulting from HIV-1 co-infection remains to be tested. It has been suggested that the increased frequency of CMV-specific T cells in ART patients may be due to abnormal responses to otherwise normal levels of subclinical CMV replication . Similar*44:03-restricted NW8-specific CD8+ T cell response deserves comment. In most individuals, >3% of CD8+ T cells, and in one individual almost 1 in 5 CD8+ T cells (19%), are specific for this single response (*44:03 might be at particular risk, not only of reduced ability to counter infections in later life, but also to combat coinfections such as HIV which also appear to induce large T cell expansions (The unusual magnitude of the HLA-Bresponse . Even byresponse , 49–51. response . If thispansions .*44:03 individuals, the CD8+ T cell response to the NW8 epitope is associated with remarkably large clonotypic expansions reminiscent of memory inflation. In the absence of longitudinal TCR analysis, this hypothesis remains to be tested formally. Nonetheless, it is clear that this repertoire is a very limited repertoire of “superdominant clones” that in the extreme can occupy almost 20% of the CD8+ T cell pool. Whether this feature is typical of memory inflation in general, or specific to CMV, will have to be determined.How this so-called memory “inflation” is established upon infection remains unclear, although it is possible that certain pathogens, like CMV, would adapt to host immunity to abrogate the contraction phase of the immune response. The composition of the “inflated” memory pool both at the cellular and clonotypic levels has also remained elusive . Our resmhc locus is the most gene-dense and the most polymorphic genetic complex known in jawed vertebrates, reflecting strong evolutionary pressure from highly mutable pathogens (mhc affects an individual's immune status by determining the collection of peptides made available to T cells during development and shaping of the TCR repertoire and also upon antigenic challenge (Intuitively, that highly prevalent pathogens such as CMV are best dealt with by TCRs which are highly prevalent across a population appears to be a beneficial trait. The athogens . Polymorhallenge . “Cat anhallenge , 57. WheThe study was conceived by AS and PG and funded by grants to AS and PG. MA, AM, MS, JR, and PO did the experimental work that was supervised by TN, SB, HK, PM, AS, and PG. The data were analyzed by MA, AM, HK, AS, and PG. The manuscript was written by MA, AM, AL, HK, PM, AS, and PG.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer EH declared a past co-authorship with one of the authors AS to the handling editor.


The Bladder Cancer Index (BCI) and Functional Assessment of Cancer Therapy-Bladder-Cystectomy (FACT-Bl-Cys) were developed to measure disease-specific health-related quality of life (HRQOL) in bladder cancer patients and patients treated with radical cystectomy, respectively. Both patient-reported outcome measures (PROMs) are frequently used in clinical practice, but are not yet validated according to the COSMIN criteria and not yet available in Dutch. Therefore, the aim of this study was to translate the BCI and FACT-Bl-Cys into Dutch and to evaluate their measurement properties according to the COSMIN criteria.The BCI and FACT-Bl-Cys were translated into Dutch using a forward-backward method, and subsequently administered at baseline (pre-operatively) and 3 months post-operatively in bladder cancer patients who received a radical cystectomy. Validity (content and construct), reliability , floor and ceiling effects, and responsiveness were assessed according to the COSMIN criteria.Forward-backward translation encountered no particular linguistic problems. In total 260 patients completed the baseline measurement, while 182 patients completed the three-month measurement. Only a ceiling effect was identified for the BCI. Hypotheses testing for construct validity was satisfying, as 67% and 92% of the hypothesized correlations were confirmed. Structural validity was moderate for both measures, as confirmatory factor analyses showed limited fit. Reliability of both PROMs was good. The intraclass correlation coefficient (ICC) of the BCI domains ranged from 0.47 to 0.93, minimal value of Cronbach’s α was 0.70, smallest detectable change on group level (SDC group) ranged from 1.9 to 8.6. The ICC of the FACT-Bl-Cys domains ranged from 0.43 to 0.83, minimal value of Cronbach’s α was 0.77, SDC group was around 1. Only the FACT-Bl-Cys total score was found to be responsive to changes in generic quality of life.The Dutch versions of the BCI and FACT-Bl-Cys were shown to be reliable and have good content validity. Structural validity was limited for both measures. Only the FACT-Bl-Cys total score was responsive to changes in generic HRQOL. Despite some limitations, both PROMs seem suitable for use in clinical practice and research.The online version of this article (10.1186/s41687-019-0149-7) contains supplementary material, which is available to authorized users. Bladder cancer (BCa) ranks ninth in worldwide cancer incidence and is one of the most expensive malignancies to manage , 2. The Due to the heterogeneous BCa population, the various treatments , and the lack of long-term follow-up data, not much is known regarding the patient burden imposed by BCa –5. SinceIn 2016, a systematic review was published in which Danna et al.  advised To assess whether both PROMs provide valuable information in clinical practice and research, it is warranted to study their measurement properties. The COnsensus-based Standards for the selection of health Measurement INstruments (COSMIN) checklist was developed to assess the quality of validation studies and can be used to design and report on these studies –22. The The BCI and FACT-Bl-Cys were translated into Dutch, using a forward–backward method according to published guidelines, involving six steps , 24. A swww.trialregister.nl) patients were asked to complete four measures . Patients that completed the three-month follow-up period were included in this substudy. RACE is a comparative effectiveness study aiming to determine the (cost-)effectiveness of robot-assisted and open radical cystectomy [As part of the RACE study , radical cystectomy performed in combination with a nephrectomy or a partial colon resection.All participants in this validation study fulfilled the inclusion criteria of the RACE study: they were 18 years or older, had an oncological indication for radical cystectomy, a histologically proven primary muscle invasive urothelial carcinoma or therapy resistant high-risk non muscle-invasive BCa , a non-metastatic tumor , they were able to complete Dutch questionnaires, and provided written informed consent. Patients were excluded from RACE when they met one of the following criteria: previous major abdominal surgery , pregnancy, morbid obesity   and the The BCI is developed to measure disease-specific HRQOL in patients with nonmuscle-invasive and muscle-invasive BCa disease of any stage. It consists of 36 items covering three domains: urinary 14 items), bowel (10 items), and sexual (12 items) , 14. Eac items, bThe FACT-Bl-Cys is developed to measure condition-specific HRQOL in patients treated with radical cystectomy. It consists of the four general FACT-General (FACT-G)  domains COSMIN recommendations were used as a guide for evaluating the measurement properties of the Dutch BCI and FACT-Bl-Cys . ValiditBecause the BCI is developed for generic BCa patients, we determined its measurement properties at baseline , with the exception of test-retest and measurement error which were based on T3 and T3 retest. The test-retest and measurement error could not be assessed at T0, because there was no possibility to distribute and complete the PROMs again within two weeks since patients received a cystectomy on short term after T0. Because the FACT-Bl-Cys is developed for patients treated with radical cystectomy, we determined its measurement properties after the cystectomy had been performed, i.e. at T3 and T3 retest..Floor and ceiling effects are considered to be present if more than 15% of respondents achieved the lowest or highest possible score, respectively , 35. We n = 20). An interview guide was used for the interviews and respondents were invited to appraise the two measurement instruments on relevance, comprehensiveness, and comprehensibility [Validity in this study was assessed by content validity and construct validity . Contentsibility . Input fConstruct validity was evaluated by structural validity and hypotheses testing. First, structural validity was assessed by confirmatory factor analyses (CFA) to confirm the previously suggested dimensionality of both PROMs . RegardiFor a CFA, COSMIN advises to use a sample size of 7 patients per item, with a minimum number of 100 patients . Since oSecond, we tested various hypotheses by analyzing the association between the BCI domains and FACT-Bl-Cys domains, using Pearson’s correlation coefficients. Coefficients were considered low (< 0.30), moderate (0.30–0.69) or high (≥0.70). If ≥75% of the set number of hypotheses were confirmed, the construct validity was considered good . Pre-speReliability was assessed by analyzing internal consistency, test-retest reliability, and measurement error .Internal consistency  of subscTest-retest reliability  was asse2o + σ2residual) [individual = 1.96*√2*SEM), using the formula: SDCindividual/√n [Measurement error  was asseesidual) , 43. Addidual/√n .mean-T0mean)/T0SD.Responsiveness should be determined in a population that shows actual change . First, Conceptual rather than literal translation was leading to preserve the original meaning of each item. There was agreement between the forward and backward translations, and no cultural adaptions regarding translation were made. Based on the cognitive interviews, two items regarding the interpretation and meaning were discussed with the authors  and FACIT representatives. First, we added one additional phrase “for men only”  to the beginning of FACT-Bl-Cys item Bl5 “I am able to maintain an erection”, as this item is specifically aimed at men. According to FACIT scoring instructions Bl5 was not included in the scoring algorithm of the scale. Therefore, adapting this item could not influence the total score. Second, the BCI items where patients are asked about their “urine loss” could be interpreted according to their own specific situation. In case of a neobladder, stoma, or own bladder questions about “urine loss” could be answered as problems with catheterizing incontinence, emptying the stoma bag or leakage from the stoma bag, or urination, respectively.In total 260 patients completed the baseline measure (T0), followed by 182 patients who completed the three-month measure post-operatively (T3). The T3 evaluation was completed at a mean of 106 days (SD:14 days). Sixty-two patients participated in the test–retest measurement (T3 retest). The T3 retest was completed at a mean of 14 days (SD:6 days) after T3. In Table Seven out of nine BCI domains showed a ceiling effect (meaning few problems), while no floor effects were observed  raised concerns by some respondents, as patients with different bladder diversions might interpret this differently . We discussed these findings with FACIT representatives, and decided that patients could respond to this item in any way they feel appropriate.The CFA was performed on the imputed datasets and the model fit was evaluated based on Comparative Fit Index (CFI), Tucker-Lewis index (TLI), Root Means Square Error of Approximation (RMSEA), and Standardized Root Mean Square Residual (SRMR). Considering structural validity for BCI, the CFA resulted in a moderate fit  were confirmed , test–retest reliability (ICC), and measurement error  are presented in Tables For BCI, Cronbach’s α of the domains ranged from 0.70 (“bowel function”) to 0.91 , which indicates a good internal consistency. All ICCs were above 0.70, except for “sexual bother” which showed an ICC of 0.47. The SEM ranged from 4.17  to 20.07 , which resulted in a SDC individual score ranging from 11.56 to 55.63. The SDC group ranged from 1.90 (urinary domain) to 8.58 . Thus for the domain “sexual bother”, an average group difference in score ≥ 8.58 cannot be attributed to only measurement error, but actual change will have taken place.For FACT-Bl-Cys, Cronbach’s α of the domains ranged from 0.77 (Bl-Cys) to 0.85 (FWB), which indicates a good internal consistency. All ICCs were above 0.70, except for SWB which showed an ICC of 0.43. The SEM ranged from 1.57 (EWB) to 3.14 (Bl-Cys domain), which resulted in a SDC individual score ranging from 4.36 to 8.69. Our SDC individual values show that, to determine a treatment effect a difference of at least 4.36 to 8.69 points between two scores from an individual patient is required to ensure that the difference is not attributed to measurement error. The SDC group ranged from 0.59 (EWB) to 1.18 (Bl-Cys). See Additional file Our total study population did not show major changes between T0 and T3 for the BCI and FACT-Bl-Cys  or a deterioration in this score of ≥10 points .Additionally, the change score between T0 and T3 of BCI and FACT-Bl-Cys in comparison to the change score of the EQ-VAS was studied. As presented in Additional file For BCI, the number of patients with an improved EQ-VAS ranged from 17 to 47, depending on the domain. We hypothesized to find a larger effect size in the improved group compared to the no-change group, but only 1 of 9 hypotheses was confirmed , “urinary function” (Cohens d: − 0.56 > − 0.45), and the sexual domain (Cohens d: − 1.10 > − 0.85). Regarding hypotheses on correlations, only 11% of all a priori hypotheses (1 of 9) were confirmed. We only found a moderate correlation for the change score of the “sexual function” domain.For FACT-Bl-Cys, the number of patients with an improved EQ-VAS ranged from 45 to 52, depending on the domain. For both effect sizes and correlations, 83% of all a priori hypotheses (5 of 6) were confirmed. Only for FACT-SWB we found a smaller effect size (Cohens d: − 0.17 < − 0.50) and a low correlation (r = 0.20).For FACT-Bl-Cys, the number of patients with a deteriorated EQ-VAS ranged from 40 to 42, depending on the domain. Regarding hypotheses on effect sizes for the deterioration group, 67% of all a priori hypotheses (4 of 6) were confirmed (see Table The aim of this study was to translate two disease-specific measures (BCI and FACT-Bl-Cys) into Dutch and to evaluate their measurement properties according to the COSMIN criteria. Both measures were found to be reliable and have good content validity, but limited structural validity. This study showed that the Dutch version of the BCI was not very responsive to changes in generic HRQOL, and showed a ceiling effect. In the Dutch version of the FACT-Bl-Cys no floor or ceiling effects were observed and the FACT-Bl-Cys total score was responsive to changes in generic HRQOL.The structural validity of the BCI was assessed as moderate, as the hypothesized domains based on the original validation  and tranAlthough experts debate on the minimum number of patients needed for a factor analysis (ranging from 100 to 300 patients) –54, COSMRegarding BCI at T0, high ceiling effects were observed for both urinary and bowel domains. The maximum scores indicate good function and no bother, which might be when patients are recently diagnosed and have minor symptoms (e.g. painless hematuria). Similar findings are reported in previous studies , 16, 17.Regarding FACT-Bl-Cys, there were uncertainties concerning the calculation of total scores, as we excluded the items BL4 ‘I am interested in sexual activity’ and BL5 ‘I am able to have and maintain an erection’, according to FACIT instructions. These items were also not included in the CFA. Previously published studies each handled these items differently , 18, 19.In comparison to previous BCI , 16, 17 Regarding construct validity of the BCI, our results confirm the suggestion that the BCI captures additional information which is not covered by generic instruments. Schmidt et al. found similar results as they reported low to moderate correlations (range: 0.16–0.48) between BCI and the SF-36 . RegardiOnly one BCI study mentioned responsiveness . In 2014We found one study that reported on longitudinal changes of FACT-Bl-Cys . AndersoAs far as we are aware, this is the first study determining the measurement properties of BCI and FACT-Bl-Cys, according to COSMIN guidelines, in a large study population. COSMIN enhances clarity and stimulates uniform usage of terminology, enables researchers to evaluate each measurement property individually, and improves comparability with other studies.n = 55) showed a mean increase of 25 points, and the population with a deteriorated EQ-VAS score (n = 89) showed a mean decrease of 50 points. Previous studies in other patient populations reported MIC values varying from 6.9 to 8.9 [This study also has some limitations. First, our study population comprised only patients treated with radical cystectomy. Therefore, we were unable to determine whether both measures will be valid and reliable in a more generic BCa population. Second, due to our study design test-retest and measurement error had to be determined at 3 months  rather than at baseline . For FACT-Bl-Cys this is an adequate moment, but for BCI this might result in a bias since this measure is originally intended for BCa patients in general. Third, to explore responsiveness we studied the correlation and effect size of change score of both measures with the change score of the EQ-VAS between T0 and T3. Although we expected that radical cystectomy impacts HRQOL, the BCI and FACT-Bl-Cys did not detect large changes between T0 and T3. Two previous studies did also not find major changes between T0 and T3 for FACT-Bl-Cys , 56. Thi9 to 8.9 –48, but Based on this study, both measures seem suitable for cross-sectional use in clinical practice and for research purposes. Consulted urologists mentioned that all topics of both measures are usually discussed with patients in clinical practice, and usage of measures could be valuable for clinicians in order to provide more personalized care. Additionally, urologists noticed that the measures seem quite long for daily practice which limits the suitability and feasibility, but removing or combining items influences validity.When using the BCI, it should be considered that the largest proportion of missing values was found in the sexual domain, which is also noticed by two previous studies , 17. DurThe Dutch versions of the BCI and FACT-Bl-Cys were shown to be reliable and have good content validity. Structural validity was limited for both measures. Only the FACT-Bl-Cys total score was responsive to changes in generic HRQOL. Despite some limitations, both measures are suitable for cross-sectional use in clinical practice and for research purposes. Exploration of a more adequate component solution and whether both PROMs are responsive when objective measures (e.g. occurrence of complications) are used as an indicator of change, needs to be further evaluated in future research.Additional file 1:Variance components of the measures. (DOCX 22 kb)Additional file 2:Change in scores between T0 (baseline) and T3 (90 days post-operative) of the measures in relation to registered complications. (DOCX 44 kb)Additional file 3:Change in scores between T0 (baseline) and T3 (90 days post-operative) of the measures in relation responsiveness properties. (DOCX 26 kb)


This study aimed to examine whether inhibition of hexokinase (HK)-II activity enhances the efficacy of sorafenib in in-vivo models of hepatocellular carcinoma (HCC), and to evaluate the prognostic implication of HK-II expression in patients with HCC. We used 3-bromopyruvate (3-BP), a HK-II inhibitor to target HK-II. The human HCC cell line was tested as both subcutaneous and orthotopic tumor xenograft models in BALB/c nu/nu mice. The prognostic role of HK-II was evaluated in data from HCC patients in The Cancer Genome Atlas (TCGA) database and validated in patients treated with sorafenib. Quantitative real-time PCR, western blot analysis, and immunohistochemical staining revealed that HK-II expression is upregulated in the presence of sorafenib. Further analysis of the endoplasmic reticulum-stress network model in two different murine HCC models showed that the introduction of additional stress by 3-BP treatment synergistically increased the in vivo/vitro efficacy of sorafenib. We found that HCC patients with increased HK-II expression in the TCGA database showed poor overall survival, and also confirmed similar results for TCGA database HCC patients who had undergone sorafenib treatment. These results suggest that HK-II is a promising therapeutic target to enhance the efficacy of sorafenib and that HK-II expression might be a prognostic factor in HCC. Hepatocellular carcinoma (HCC) is one of the leading worldwide causes of cancer-related deaths and is the second most common cancer in men worldwide . BecauseSeveral studies have demonstrated that a variety of mechanisms are involved in sorafenib resistance, which includes increased MAPK14 activity , activatWe hypothesized that upregulated HK-II expression not only leads to the reduced efficacy of sorafenib but is also a predictor of tumor resistance to sorafenib. In order to investigate whether or not the upregulation of HK-II expression affects the efficacy of sorafenib, we analyzed the activity of sorafenib in HK-II overexpressing human HCC cell lines in in-vivo mouse models. In addition, we retrospectively evaluated whether or not HK-II expression in the tumor tissues of sorafenib-treated patients with HCC was a prognostic indicator.In this study, we show that upregulated HK-II reduced sorafenib-induced apoptotic cell death and that 3-bromopyruvate (3-BP), an HK-II inhibitor , attenuaWe first investigated whether 3-BP combined with sorafenib inhibited proliferation of HCC cells in vitro. For this purpose, human HCC cell lines, SNU-761  and Huh-p = 0.032). We assessed whether the increased glycolysis after sorafenib was HK-II dependent and found that increased glycolysis after sorafenib was reversed by 3-BP, an HK-II inhibitor (p = 0.024)  . Taken t3)). As shown in p = 0.048). Immunohistochemical analysis showed that HK-II expression was significantly increased in tumors after sorafenib treatment . Consistent with our observations of the subcutaneous tumor model, imaging analysis showed that sorafenib alone or 3-BP alone inhibited progression of the liver tumors. However, the combination treatment of 3-BP + sorafenib showed the strongest anti-tumor effect . At the end of the last follow-up, 68 patients had died of the disease, and the proportion of patients who died of the disease showed high HK-II expression levels in their tumors than those were alive. The median OS rates were 56.5 months (interquartile range (IQR) = 12.2–41.4) and 84.7 months (IQR = 13.8–45.6), for the HK-II-high and HK-II-low patients, respectively   .Based on our results, we postulated that HK-II expression and activity are correlated with sorafenib resistance.n = 94) demonstrated that HK-II expression was increased in the tumor tissue of 72 cases (76.6%), whereas 22 cases (23.4%) showed decreased HK-II expression compared to adjacent nonmalignant liver tissue . High HK-II expression was independently associated with shorter TTP (adjusted HR (aHR) 1.909; 95% CI, 1.086–3.342; p = 0.026) after adjustment for age  after adjustment for Child–Pugh score, tumor number, and lymph node involvement  anti-tumor effect provided by 3-BP + sorafenib. This synergistic effect was attributed to the following two mechanisms: (i) the inhibition of aerobic glycolysis, which is enhanced after sorafenib exposure, resulting in energy depletion and apoptosis; and (ii) the promotion of ER stress due to sorafenib, also resulting in augmented apoptosis. Furthermore, the upregulation of HK-II expression in HCC tissue predicted poor survival of patients from The Cancer Genome Atlas (TCGA) database. We also found that HK-II expression in the tumors of HCC patients predicted resistance to sorafenib.Although sorafenib is the only approved systemic chemotherapy for HCC, it has only shown modest results for OS . Due to Inhibitors of aerobic glycolysis via the Warburg Effect have been reported to be effective, and some of the inhibitors are under clinical study . When aeIncreased ER stress is one of the recently observed mechanisms of the antitumor actions of sorafenib. Previous studies have suggested that sorafenib increases ER stress, which leads to caspase-3 induced cell death . HexokinIn our patient cohort, HK-II expression was significantly correlated with OS and TTP in patients who had undergone sorafenib treatment. However, we could not find an association between HK-II expression and different stage or tumor grade. Additional studies of other patient cohorts are needed to verify our findings. The relationship between other known resistance factors such as HIF-1α and VEGFR, and OS and TTP should also be investigated ,33. In aIn conclusion, HK-II is a useful therapeutic target that can lead to enhancement of the efficacy of sorafenib treatment. HK-II expression might also be useful as a marker that predicts sensitivity to sorafenib and clinical outcomes.2. The culture medium was replaced every three days.The SNU-761 human HCC cell line  was main® 2000  with the pMSCV/luciferase vector (5 μg), a gag-pol vector (5 μg), and an envelope vector (5 μg). Supernatants were collected at 48 h posttransfection and filtered with 0.45-μm polyvinylidene difluoride (PVDF) filters . Virus supernatants were tittered and stored at −80 °C until required. For viral transduction of cancer cells, virus containing medium was added to target cells and polybrene  or protamine sulfate was also added to the target cells. Luciferase-expressing cancer cells were pooled after puromycin selection.A lentivirus was used for the stable transduction of the firefly luciferase-expressing SNU-761 cells. Stock virus was generated in the 293FT cell line, which was co-transfected via LipofectamineThe agent 3-BP was obtained from Sigma-Aldrich, Inc. . Sorafenib was purchased from LC laboratories .Lactate production was used as a surrogate marker of glycolysis . The SNUThe expression of genes induced by ER stress, GADD34 and GADD153, was monitored ,19. Tota3VO4; 1 mM NaF) and centrifuged at 14,000× g for 10 min at 4 °C. Samples were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, exposed to appropriate primary antibodies, and incubated with peroxidase-conjugated secondary antibodies . Bound antibodies were visualized via the use of a chemiluminescent substrate  and exposed to Kodak X-OMAT film. The primary antibodies used in this study were as follows: mouse anti-phospho-eukaryotic initiation factor 2α (eIF2α) and anti-actin were obtained from Santa Cruz Biotechnology, Inc. ; rabbit anti-caspase-9, rabbit anti-caspase-7 and mouse anti-phospho-JNK were obtained from Cell Signaling Technology, Inc. .Immunoblot assays were performed in the same manner as described in previous studies ,35. Brie6 cells of SNU-761 in 40 μL of Matrigel were injected into the left flank of 7-week-old mice. Mice with 150- to 200-mm3 tumors were randomly assigned to 4 groups (8 mice per group). Tumor volume was measured by calipers every week and calculated by the following formula V = (length [mm] × width [mm]2) × 0.5 [mm]3. Orthotropic tumors were established by injecting 2.5 × 106 cells ofluciferase-expressingSNU-761 in 40 μL of PBS under the subcapsular left liver lobe of anesthetized mice after subcostal mini laparotomy. Six days after the procedure, the mice were randomized into the following 4 groups  based on the adequate luciferase signals as measured byIVIS 100 . An IVIS100 imaging system  was used for the acquisition of bioluminescent images. D-luciferin potassium salt  was used as a substrate for luciferase, and 100 μL of the D-luciferin solution was injected intraperitoneally into the mice. Bioluminescent images were serially acquired until maximum signals were obtained. To quantify emitted light, regions of interest (ROI) were drawn over the regions of the tumors. Total photon flux was expressed as photons per cm2 per second per steradian (p/cm2/s/sr). Drugs  were injected intraperitoneally every 5 days for 4 weeks [Male BALB/c nu/nu mice were purchased from Orient-Bio . All animal experiments were approved by the Institutional Animal Care and Use Committee at Seoul National University Hospital (SNUH-IACUC). To establish subcutaneous tumors, 1 × 10 4 weeks . Biolumihttp://genome-cancer.ucsc.edu/). The study included patients who were histopathologically diagnosed with HCC, had not undergone pretreatment, and had complete data on OS [Hexokinase-II mRNA expression levels were retrieved from the TCGA RNA sequence database . The Mann–Whitney U was used to determine differences between groups. For the retrospective patient study, the TTP was calculated from the first day of sorafenib administration to the date of progression. Overall survival was calculated from the date of first administration of sorafenib to the date of death or last contact. Conventional clinical factors at the time of entry into the study and immunopositivity for HK-II were analyzed to identify variables that affected survival as determined by the Kaplan–Meier method and compared by the log-rank test. Stepwise multivariate analysis was performed using the Cox proportional hazards model to identify independent variables that affected survival. Factors found to be significantly related to outcome by univariate analysis were included in the multivariate analysis. For statistical analysis, SPSS version 21.0 for Windows  was used. Where The study protocol was approved by the Institutional Review Board of the hospital , and also conformed to the ethical guidelines of the World Medical Association Declaration of Helsinki. The biospecimens for this study were provided by the Seoul National University Hospital Human Biobank, a member of the Korea Biobank Network, which is supported by the Ministry of Health and Welfare. All samples derived from the National Biobank of Korea were obtained with informed consent under institutional review board-approved protocols.